The long-term objective of this research is the development of rational methods for the design and improvement of membrane-penetrating, amphipathic peptides that are antibiotic or cytolytic, or can carry other toxic molecules as cargo into cells. Critical knowledge in reaching this objective is the determination of the mechanism(s) of membrane penetration by these peptides. An important step in obtaining this information is the proposal in this application of an experimental kinetic study, combined with a global theoretical analysis that we have developed, of membrane penetration by a set of known cytotoxic or antibacterial amphipathic peptides. Peptides in this class are known to exhibit considerable target specificity which appears to derive from the interaction of the peptides with the lipid bilayer of the target cell membrane without the intervention of protein receptors. The mechanism of this widespread biological defense system is presently not understood. Four peptides that differ in length (from 14 to 37 amino acids), charge (0 to +6), and specificity (antibiotic, antifungal, or hemolytic), were selected to test a set of current models of peptide mechanisms. In addition, the chimeric construct transportan TP10 will also be examined in order to determine its mechanism and usefulness as a drug delivery vehicle. Transportans have been shown to be able to transport even large proteins, such as green fluorescent protein, into cells. It will be determined whether and how attachment of cargo modifies the mechanism of the peptide action. In order to be useful as vehicles to transport cargo into cells, peptides must translocate into the interior of vesicles without getting permanently inserted into the bilayer. This study will indicate which of the types of peptides examined is the best template to build such a peptide vehicle. Cargo-carrying peptides can be used to transport drugs into eukaryotic cells or antibiotics into bacterial cells. Surmounting cellular barriers, including intracellular compartments, is a major difficulty in the use of antibiotics.